The TREM2 H157Y Variant Influences Microglial Phagocytosis, Polarization, and Inflammatory Cytokine Release

Previously, we reported that H157Y, a rare coding variant on exon 3 of the triggering receptor expressed on myeloid cells 2 gene (TREM2), was associated with Alzheimer’s disease (AD) risk in a Han Chinese population. To date, how this variant increases AD risk has remained unclear. In this study, using CRISPR-Cas9-engineered BV2 microglia, we tried to investigate the influence of the Trem2 H157Y variant on AD-related microglial functions. For the first time, we revealed that the Trem2 H157Y variant inhibits microglial phagocytosis of amyloid-β, promotes M1-type polarization of microglia, and facilitates microglial release of inflammatory cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor-α. These findings provide new insights into the cellular mechanisms by which the TREM2 H157Y variant elevates the risk of AD.


Introduction
Currently, Alzheimer's disease (AD) is the most common type of neurodegenerative disorder in the elderly population [1]. It is characterized by progressive decline in cognition, including memory, decision making, and linguistic functions [2]. The pathological features of AD include amyloid-β (Aβ) deposition, tau hyperphosphorylation, loss of neurons and synapses, and neuroinflammation [3]. However, the etiology and pathogenesis of AD remain elusive.
Accumulating evidence suggests that activation of microglia contributes to the pathogenesis of AD [4]. On the one hand, activated microglia participate in the phagocytosis of Aβ, thus preventing amyloid plaque formation [5]. On the other hand, long-term activation of microglia leads to release of inflammatory cytokines, which subsequently causes bystander neuronal and synaptic injuries [6]. Several lines of evidence have indicated that multiple important functions of microglia, including phagocytosis, polarization, and release of cytokines, are tightly regulated by several immune receptors, such as triggering receptor expressed on myeloid cells 2 (TREM2) [7][8][9][10][11]. TREM2 is located on human chromosome 6p21, a hot zone linked with AD susceptibility [12,13]. We previously revealed that H157Y, a coding variant on exon 3 of TREM2, was associated with AD risk in a Han Chinese population [14,15]. To date, how this variant increases AD risk has not been fully understood.
In this study, using CRISPR-Cas9-engineered BV2 microglia, we tried to investigate the influence of the Trem2 H157Y variant on AD-related microglial functions. For the first time, we revealed that the Trem2 H157Y variant inhibits microglial phagocytosis of Aβ, promotes M1-type polarization of microglia, and facilitates microglial release of Brain Sci. 2023, 13, 642 2 of 8 inflammatory cytokines. These findings provide insights into the cellular mechanism by which the Trem2 H157Y variant elevates the risk of AD.

CRISPR-Cas9-Mediated Trem2 H157Y Variant Knock-In
BV2 mouse microglia with the Trem2 H157Y variant were generated using CRISPR-Cas9 technology. Based on the genomic sequence of mouse Trem2, gRNA was designed to target the regions near the variant site [16]. Cleavage efficiency was estimated by sequencing trace analysis with online tools [17,18]. A donor template containing the wild type (WT) C or variant T allele on gRNA recognition sites was designed. Trem2 was mutated by co-transfection of plasmids containing the gRNA and Cas9. The transfected BV2 microglia were plated in 96-well plates by limit dilution to generate isogenic single clones. The clones were selected from wells and screened by restriction endonuclease digestion and DNA sequencing.

ELISA
ELISA was carried out as described previously [20]. The culture medium of BV2 microglia was collected and centrifuged at 1000 × g for 15 min to remove cellular debris. The levels of the inflammatory cytokines, including interleukin (IL)-1β (Abcam, Inc., Boston, MA, USA), IL-6 (Abcam, Inc., Boston, MA, USA), and tumor necrosis factor (TNF)-α (Abcam, Inc., Boston, MA, USA), were detected by specific detection kits according to the manufacturer's protocols.

Statistical Analysis
The statistical analysis was carried out using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA, USA), as described previously [20]. One-way ANOVA followed by Tukey's post hoc test was used to analyze differences among three groups. The Student's t-test was employed for comparisons between two groups. Data are expressed as the means ± SDs. p < 0.05 was considered statistically significant.

The Trem2 H157Y Variant Inhibits Microglial Phagocytosis of Aβ
Given that microglial phagocytosis and degradation of Aβ plays a crucial role in AD progression [5], we employed an in vitro assay to investigate the influence of the Trem2 H157Y variant on microglial Aβ phagocytosis and degradation. As revealed in Figure 1A, BV2 microglia with the Trem2 H157Y variant showed a 51.1% decrease in internalized Aβ 1-42 levels when compared with WT Trem2 BV2 microglia. It is worth noting that no significant difference was observed in Aβ 1-42 degradation indexes between BV2 microglia with the Trem2 H157Y variant and WT Trem2 BV2 microglia ( Figure 1C).

ELISA
ELISA was carried out as described previously [20]. The culture medium of BV croglia was collected and centrifuged at 1000 × g for 15 min to remove cellular debri levels of the inflammatory cytokines, including interleukin (IL)-1β (Abcam, Inc., B MA, USA), IL-6 (Abcam, Inc., Boston, MA, USA), and tumor necrosis factor (T (Abcam, Inc., Boston, MA, USA), were detected by specific detection kits according manufacturer's protocols.

Statistical Analysis
The statistical analysis was carried out using GraphPad Prism software (Grap Software, Inc., San Diego, CA, USA), as described previously [20]. One-way ANOV lowed by Tukey's post hoc test was used to analyze differences among three group Student's t-test was employed for comparisons between two groups. Data are expr as the means ± SDs. p < 0.05 was considered statistically significant.

The Trem2 H157Y Variant Inhibits Microglial Phagocytosis of Aβ
Given that microglial phagocytosis and degradation of Aβ plays a crucial role progression [5], we employed an in vitro assay to investigate the influence of the H157Y variant on microglial Aβ phagocytosis and degradation. As revealed in Figu BV2 microglia with the Trem2 H157Y variant showed a 51.1% decrease in internalize The index of Aβ1-42 degradation was expressed as the r the total amount of internalized Aβ1-42 (t = 6 h) to remaining Aβ1-42 (t = 12 h). A higher degra index indicates a better capacity for degradation of Aβ1-42. Data were analyzed by one-way A followed by Tukey's post hoc test. Columns represent means ± SDs (n = 3, performed in tripl * p < 0.05 versus WT Trem2 BV2 microglia.

The Trem2 H157Y Variant Promotes M1-Type Polarization of Microglia
After stimulation, microglia showed either pro-inflammatory status (M1-type polarization) or anti-inflammatory status (M2-type polarization) [23]. To explore the influence of the Trem2 H157Y variant on microglial polarization, BV2 microglia were stimulated with 100 ng/mL LPS. As revealed in Figure 2A, B, LPS stimulation increased the levels of iNOS, a marker of M1-type polarization (p < 0.05) [24], in WT Trem2 BV2 microglia. Contrastingly, BV2 microglia with the Trem2 H157Y variant exhibited significantly higher Brain Sci. 2023, 13, 642 4 of 8 levels of iNOS after LPS stimulation. As indicated in Figure 2A, C, D, LPS stimulation increased the protein levels of ARG1 and CD206, two markers of M2-type polarization [25], in WT Trem2 BV2 microglia, but these increments did not reach statistical significance (p > 0.05). In contrast, BV2 microglia with the Trem2 H157Y variant showed reduced protein levels of ARG1 and CD206 following LPS stimulation.

FOR PEER REVIEW 4 of 8
Contrastingly, BV2 microglia with the Trem2 H157Y variant exhibited significantly higher levels of iNOS after LPS stimulation. As indicated in Figure 2A, C, D, LPS stimulation increased the protein levels of ARG1 and CD206, two markers of M2-type polarization [25], in WT Trem2 BV2 microglia, but these increments did not reach statistical significance (p > 0.05). In contrast, BV2 microglia with the Trem2 H157Y variant showed reduced protein levels of ARG1 and CD206 following LPS stimulation.

The Trem2 H157Y Variant Facilitates Microglial Release of Inflammatory Cytokines
Afterward, we investigated the influence of the Trem2 H157Y variant on microglial inflammatory cytokine release. Stimulating WT Trem2 BV2 microglia with LPS led to release of IL-1β, IL-6, and TNF-α into the culture medium. As revealed in Figure 3A-C, BV2 microglia with the Trem2 H157Y variant released more inflammatory cytokines, including IL-1β, IL-6, and TNF-α, than WT Trem2 BV2 microglia after LPS stimulation. , and CD206 protein levels. Data were normalized to β-actin. Data were analyzed by one-way ANOVA followed by Tukey's post hoc test. Columns represent means ± SDs (n = 3, performed in triplicates). * p < 0.05 versus WT Trem2 BV2 microglia without LPS stimulation; # p < 0.05 versus LPS-stimulated WT Trem2 BV2 microglia.

The Trem2 H157Y Variant Facilitates Microglial Release of Inflammatory Cytokines
Afterward, we investigated the influence of the Trem2 H157Y variant on microglial inflammatory cytokine release. Stimulating WT Trem2 BV2 microglia with LPS led to release of IL-1β, IL-6, and TNF-α into the culture medium. As revealed in Figure 3A-C, BV2 microglia with the Trem2 H157Y variant released more inflammatory cytokines, including IL-1β, IL-6, and TNF-α, than WT Trem2 BV2 microglia after LPS stimulation.

Discussion
TREM2 is a newly identified susceptibility gene for AD [26]. In Caucasians, coding variant, R47H, was reported to increase the risk of AD with an odds ratio [27,28]. Meanwhile, we identified that another rare coding variant H157Y substa elevated AD risk in a Han Chinese population [14]. To date, how the TREM2 H157 ant increases AD risk has not been fully understood. Human TREM2 encodes a 230acid type I transmembrane receptor, which is exclusively expressed in microglia brain [29]. It contains an Ig-like V-type domain, a stalk region, a transmembrane do and a short cytoplasmic tail [30]. An increasing amount of evidence suggested th length TREM2 participated in AD progression via facilitated microglial phagocyt Aβ and suppressed M1-type polarization of microglia [22,31]. In this study, we hav vided the first evidence that the Trem2 H157Y variant inhibits microglial Aβ phagoc This finding was supported by a recent study from Schlepckow and colleagues sh that the TREM2 H157Y variant impaired phagocytosis of E.coli pHrodo parti HEK293 cells [32]. Moreover, in the current study, we observed that the M1-type zation marker iNOS was increased while the M2-type polarization markers ARG CD206 were reduced in BV2 microglia with the Trem2 H157Y variant. To our know this is the first study reporting that the Trem2 H157Y variant induced microglial M polarization. Under physiological conditions, a disintegrin and metalloprot (ADAM) 10/17 cleaves the full-length TREM2 protein at the H157-S158 site [ HEK293 cells, the TREM2 H157Y variant was revealed to facilitate ADAM10/17 cle and thus reduce membrane-associated full-length TREM2 protein levels [32]. In v the above evidence, the M1-type microglial polarization and impaired microgl phagocytosis caused by the H157Y variant might be ascribed to the reduction i length TREM2 protein levels on the surface of microglia.
Another interesting finding of this study was that the Trem2 H157Y variant pro microglial release of inflammatory cytokines. The TREM2 H157Y variant was repo enhance sTREM2 production via enhanced ADAM10/17 cleavage [33]. As a bioactiv ment of full-length TREM2 protein, sTREM2 was reported to promote microglial i matory cytokine release and subsequently induce neuroinflammation [34]. Therefo elevated inflammatory cytokine release caused by the Trem2 H157Y variant might tributed to the enhanced sTREM2 production. However, our findings appeared to c dict those of a recent study by Qiao and colleagues [35], as they found that the H157Y variant downregulated neuroinflammation-related genes in Trem2 H157Y in 5xFAD transgenic mice. It should be noted that our experiments were conducte cellular inflammation model induced by LPS, whilst Qiao et al. employed an a

Discussion
TREM2 is a newly identified susceptibility gene for AD [26]. In Caucasians, a rare coding variant, R47H, was reported to increase the risk of AD with an odds ratio of 4.7 [27,28]. Meanwhile, we identified that another rare coding variant H157Y substantially elevated AD risk in a Han Chinese population [14]. To date, how the TREM2 H157Y variant increases AD risk has not been fully understood. Human TREM2 encodes a 230-amino acid type I transmembrane receptor, which is exclusively expressed in microglia in the brain [29]. It contains an Ig-like V-type domain, a stalk region, a transmembrane domain, and a short cytoplasmic tail [30]. An increasing amount of evidence suggested that fulllength TREM2 participated in AD progression via facilitated microglial phagocytosis of Aβ and suppressed M1-type polarization of microglia [22,31]. In this study, we have provided the first evidence that the Trem2 H157Y variant inhibits microglial Aβ phagocytosis. This finding was supported by a recent study from Schlepckow and colleagues showing that the TREM2 H157Y variant impaired phagocytosis of E.coli pHrodo particles in HEK293 cells [32]. Moreover, in the current study, we observed that the M1-type polarization marker iNOS was increased while the M2-type polarization markers ARG1 and CD206 were reduced in BV2 microglia with the Trem2 H157Y variant. To our knowledge, this is the first study reporting that the Trem2 H157Y variant induced microglial M1-type polarization. Under physiological conditions, a disintegrin and metalloproteinase (ADAM) 10/17 cleaves the full-length TREM2 protein at the H157-S158 site [33]. In HEK293 cells, the TREM2 H157Y variant was revealed to facilitate ADAM10/17 cleavage and thus reduce membrane-associated full-length TREM2 protein levels [32]. In view of the above evidence, the M1-type microglial polarization and impaired microglial Aβ phagocytosis caused by the H157Y variant might be ascribed to the reduction in full-length TREM2 protein levels on the surface of microglia.
Another interesting finding of this study was that the Trem2 H157Y variant promoted microglial release of inflammatory cytokines. The TREM2 H157Y variant was reported to enhance sTREM2 production via enhanced ADAM10/17 cleavage [33]. As a bioactive fragment of full-length TREM2 protein, sTREM2 was reported to promote microglial inflammatory cytokine release and subsequently induce neuroinflammation [34]. Therefore, the elevated inflammatory cytokine release caused by the Trem2 H157Y variant might be attributed to the enhanced sTREM2 production. However, our findings appeared to contradict those of a recent study by Qiao and colleagues [35], as they found that the Trem2 H157Y variant downregulated neuroinflammation-related genes in Trem2 H157Y knock-in 5xFAD transgenic mice. It should be noted that our experiments were conducted in a cellular inflammation model induced by LPS, whilst Qiao et al. employed an animal model of amyloid pathology to validate the function of the Trem2 H157Y variant [35]. The pathophysiological bases underlying these two models are quite different, which may account for these opposite observations. This study also had some limitations. First, the impacts of the H157Y variant on the TREM2 downstream signaling pathway were not investigated in this study. In the future, the main components of the TREM2 downstream signaling pathway, such as TYROBP and β-catenin in microglia with the Trem2 H157Y variant, should be assessed [36][37][38]. Second, in this study, the functions of the H157Y variant were investigated using mouse BV2 microglial cells. Although TREM2 protein seemed to be conserved across mammals, our findings should be further confirmed using human microglia.

Conclusions
Summarily, in the current study, using CRISPR-Cas9-engineered BV2 microglia, we provide the first evidence that the Trem2 H157Y variant inhibits microglial phagocytosis of Aβ, promotes M1-type polarization of microglia, and facilitates microglial release of inflammatory cytokines. These findings provide new insights into the cellular mechanisms by which the TREM2 H157Y variant elevates the risk of AD.